(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
(19) World Intellectual Property Organization
International Bureau
(43) International Publication Date
10 July 2003 (10.07.2003)
PCT
(10) International Publication Number
WO 03/056345 Al
(51) International Patent Classification 7 : G01N 35/10
(21) International Application Number: PCT/KR02/00703
(22) International Filing Date: 17 April 2002 (17.04.2002)
(25) Filing Language: English
(26) Publication Language: English
(30) Priority Data:
2001/84331
24 December 2001 (24.12.2001) KR
(71) Applicant (for all designated States except US): I-SENS,
INC. [KR/KR]; 447-1 Wolgye-dong, Nowon-ku, 139-701
Seoul (KR).
(72) Inventors; and
(75) Inventors/Applicants (for US only): CUI, Gang
[CN/CN]; 19 zhu Minghui-wei, Xinxing-je, Yanji,
133000 Jilin (CN). KIM, Ju-Yong [KR/KR]; 528-6
Wooman-dong, Paldal-gu, Suwon, 442-190 Kyoungki
(KR). KIM, Moon-Hwan [KR/KR] ; Ma-dong 501-Ho
Dongwon Apt., 363 Gogangbon-dong, Ojung-gu, Buchon,
421-190 Kyoungki (KR). UHM, Jung-Hee [KR/KR] ;
174-29 Jangwi 3-dong, Seongbuk-gu, 136-143 Seoul
(KR). NAM, Hakhyun [KR/KR]; #101-206 Hanyang
Apt, 133-1, Beon 3-dong, Kangbuk-ku, 142-063 Seoul
(KR). CHA, Geun-Sig [KR/KR]; #4-207 Cheongwoon
Apt., San 4-25, Cheongwoon-dong, Chongro-ku, 110-030
Seoul (KR).
(74) Agent: LEE, Won-Hee; 8th Fl., Sung-ji Heights II 642-16,
Yoksam-dong, Kangnam-ku, Seoul 135-080 (KR).
(81) Designated States (national): AE, AG, AL, AM, AT, AU,
AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU,
CZ, DE, DK, DM, DZ, EC, EE, ES, K, GB, GD, GE, GH,
GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KZ, LC, LK,
LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX,
MZ, NO, NZ, OM, PH, PL, PT, RO, RU, SD, SE, SG, SI,
SK, SL, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VN,
YU, ZA, ZM, ZW.
[Continued on next page]
(54) Title: ELECTROCHEMICAL BIOSENSORS
300
IT)
IT)
400
(57) Abstract: There is provided electrochemical biosensors with a sample introducing part, comprising a sample introducing pas-
sage, an air discharge passage, and a void. The sample introducing passage communicates with the air discharge passage, and the
void is formed at the point of communication. Also, disclosed is the electrochemical biosensor with the said sample introducing part
and a fluidity determining electrode.
WO 03/056345 Al 11 111 « II HUH I II 1 11 111 111 111 11 Nil
(84) Designated States (regional): ARIPO patent (GH, GM,
KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM, ZW),
Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
European patent (AT, BE, CH, CY, DE, DK, ES, FI, FR,
GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OAPI patent
(BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, ML, MR,
NE, SN, TD, TG).
Published:
with international search report
For two-letter codes and other abbreviations, refer to the "Guid-
ance Notes on Codes and Abbreviations" appearing at the begin-
ning of each regular issue of the PCT Gazette.
WO 03/056345
PCT/KR02/00703
ELECTROCHEMICAL BIOSENSORS
FIELD OF THE INVENTION
The present invention relates to electrochemical
5 biosensors. More particularly , the present invention relates
to electrochemical biosensors with an enhanced sample
introducing part; the sample introducing part comprising a
sample introducing passage, an air discharge passage, and a
void, wherein the sample introducing passage communicates with
10 the air discharge passage and wherein the void is formed at
the point of communication. The present invention also
provides a method for determining the fluidity of blood
samples utilizing the said sample introducing part.
15 BACKGROUND OF THE INVENTION
Periodic monitoring of blood glucose levels is needed
for the diagnosis and prophylaxis of diabetes mellitus . The
conventional analyzers for detecting the level of glucose in
20 blood are strip-type analyzers based on either a color imetric
method or an electrochemical method.
The colorimetric method depends on a glucose oxidase-
colorimetric reaction :
25
glucose + 0 2 -> gluconic acid + H 2 0 2 (catalyst: glucose oxidase)
H 2 0 2 + dye -> product (catalyst: peroxidase)
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As shown in the reaction, glucose reacts with oxygen and
is oxidized to gluconic acid and hydrogen peroxide in the
presence of glucose oxidase. With the aid of peroxidase, the
hydrogen peroxide is then reduced to water while oxydizing
5 chromophoric oxygen receptor. This reaction result in color
change proportional to the level of glucose in blood.
This colorimetric method, however, requires precise care,
because the change of the color (or intensity) depends on the
10 degree of sample transport and sample pre-treatment , quantity
of sample, reaction time and coloration time. In addition,
blood coagulation or the presence of interfering materials
(for example, uric acid, ascorbic acid, and bilirubin) may
disturb the colorimetric analysis.
15 Electrochemical method may avoid the above problems,
providing high selectivity and sensitivity. For example, an
electrochemical biosensor enables samples to be introduced
without pre-treatment, even if the samples are turbid, and
makes it possible to accurately analyze the level of glucose
20 within a short period of time.
Both colorimetric and electrochemical methods which use
oxygen as an electron transfer mediator are called as the
first-generation biosensor. The second-generation
electrochemical adopt organometallic compounds (e.g., Fe, Os,
25 Ru containing derivatives) , quinones, quinone derivatives,
organic conducting salts or viologen as an electron transfer
mediator. The second-generation electrochemical sensors are
based on the reaction:
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glucose + GOx_ F ad -> gluconic acid + GOx. FA dh2
GOX. F ADH2 + M ox -> GOxfad + M rec r
5 (wherein, GOx represents glucose oxidase; GOx_ FAD and GOx_ FADH 2
represent an oxidized state and a reduced state of glucose
oxidase, respectively; and, M ox and M re d denote the oxydized
and reduced electron transfer mediator, respectively. )
10 As shown in the reaction, glucose is oxidized to
gluconic acid by reducing G0x_ F ad to G0x_ FA dh2 • The reduced
glucose oxidase transfers an electron (s) to the electron
transfer mediator M ox and then returns to the initial state.
During this reaction, the redox current thus generated is
15 measured at the surface of the electrode.
The electrochemical biosensor strip comprises: a) at
least one substrate on which an electrode system (a working
electrode, an auxiliary electrode and/or reference electrode)
20 is printed, b) an oxidase and an electron transfer mediator
immobilized on the electrode system, and c) a sample
introducing part. The electrochemical biosensor strip may be
classified into four types: (1) a flat-type biosensor in which
a working electrode and an auxiliary electrode (or a reference
25 electrode) are printed on the same base substrate; (2) a
converse-type biosensor in which a working electrode and an
auxiliary electrode are facing each other and; (3) a
differential flat-type biosensor; and (4) a differential
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converse-type biosensor. '
Most commercially available biosensors have a sample
introducing part that might be classified as either an i-type
5 or a horizontal line-type.
The i-type sample introducing part comprises base
substrate, a thin film spacer (typically, 100 - 500 jam) with
U-shaped cut-out portion, and the cover plate with a vent hole
for discharging the air. The vent hole may be formed at the
10 base plate as well. This type of biosensor provides a rapid
introduction of liquid sample through the i-type capillary,
but suffers from the disadvantages that the amount of the
sample introduced is not precisely controlled because the U-
shaped channel is often over filled or under filled around the
15 vent hole; the filling of the sample channel significantly
depends on the fluidity of blood which varies largely with the
hematocrit level. Another disadvantage of i-type is that
improper handling of the strip easily contaminates the user
with the blood squeezed through the vent hole.
20
The horizontal line-type sample introducing part is
formed by the spacer arranged to form a narrow flow channel
crossing the strip between the base and cover substrates; the
sample is introduced through the inlet formed on one lateral
25 side, while an air within the space is discharged through the
outlet formed on the other lateral side. This type of
biosensor also suffers from the disadvantage that a sample
should be introduced laterally, often forcing the user to
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place the strip in an awkward position over the sampling area.
Therefore, according to the first aspect of the present
invention, there is provided an electrochemical biosensor
5 equipped with a sample introducing part that enables a rapid
introduction of a blood sample at the tip of the strip in
accurate amount for electrochemical determination.
Human blood contains solid particles (hematocrits) such
10 as erythrocytes, white cells and other proteins, which can be
separated from the plasma. These particles change the
fluidity and electrical conductivity of blood. It is noted
that the sample is introduced in different speed to the
capillary channel of a biosensor strip, and the sample
15 filling time is a function of hematocrit level.
Therefore, according to the second aspect of present
invention, there is provided an electrochemical biosensor
equipped with a fluidity determining electrode that measures
20 the sample fill-up time in the capillary, and a method to
correct the values with respect to those at a given hematocrit
level .
SUMMARY OF THE INVENTION
25
An object of the present invention is to provide an
electrochemical biosensor with a sample introducing part that
enables rapid and accurate introduction of physiological
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sample,, without any pre- treatment of a blood sample .
Another object of the present invention is to provide an
electrochemical biosensor equipped with a sample fluidity
determining electrode, wherein the influences of fluidity
5 modifying components are effectively corrected. The fluidity
determining electrode also discriminates abnormal samples,
such as the blood samples with unusual viscosity (too high or
too low compared to that of normal human blood) or the
samples containing air bubbles (US5, 284 , 658) .
10
These and other objects can be accomplished by providing
the sample introducing part comprising a sample introducing
passage, an air discharge passage, and a void, wherein the
sample introducing passage communicates with the air discharge
15 passage and wherein the void is formed at the point of
communication, and wherein the void may be utilized further to
place a fluidity determining electrode.
BRIEF DESCRIPTION OF THE DRAWINGS
20
The application of the preferred embodiments of the
present invention is best understood with reference to the
accompanying drawings, in which like reference numerals are
used for like and corresponding parts, wherein:
25 Fig. 1 is an exploded perspective view, which
illustrates an electrochemical biosensor with a sample
introducing part according to the present invention;
Fig. 2 is an exploded perspective view showing a flat
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type biosensor, in accordance with a first embodiment of the
present invention;
Fig. 3 is an exploded perspective view showing a
converse type biosensor, in accordance with a second
5 embodiment of the" present invention;
Fig. 4 is an exploded perspective view showing
differential flat type biosensor, in accordance with a third
embodiment of the present invention;
Fig. 5 is an exploded perspective view showing a
10 differential converse type biosensor, in accordance with a
fourth embodiment of the present inventions-
Fig. 6 is an exploded perspective view, which
illustrates an electrochemical biosensor with a sample
introducing part and a fluidity detection electrode according
15 to the present invention;
Fig. 7 is the graph that shows the influence of various
interfering materials on a converse type glucose sensor;
a: Glucose
b: Glucose + Acetoaminophen (660 fxM)
20 c: Glucose + Ascorbic acid (570 \xM)
d: Glucose + Uric acid (916 \M)
Fig. 8 is a graph showing a calibration curve of a
converse type glucose sensor, for sensitivity to glucose
standard solution; and
25 Fig. 9 is a graph showing dynamic curves, obtained by a
chronoamperometric method, of a converse type glucose sensor,
for glucose standard solutions.
Fig. 10 is a graph that illustrates the relationship
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between the sample fluidity (as a function of time) and the
hematocrit level .
DETAILED DESCRIPTION OF THE INVENTION
5
With reference to Fig. 1, an electrochemical biosensor
comprises a spacer 200 and a lower substrate 400 (base) and an
upper substrate (cover) 300 for forming the electrochemical
sensors and sample introducing channel. Formed into one end
10 of the spacer 200 is a sample introducing passage 101, an air
discharge passage 102, and a void 103. Notably, the sample
introducing passage 101 communicates with the air discharging
passage 102 in a roughly perpendicular manner, and the void
103 is formed at the point of communication. Taken as a whole,
15 the sample introducing passage 101, air discharge passage 102,
and void 103 constitute a sample introducing part 100.
The sample introducing passage 101 is a passage capable
of introducing the sample into the biosensor, and the air
discharge passage 102 is a passage for air. Due to capillary
20 action, a sample to be tested is introduced into the sample
introducing part 100 and an air is discharged through the air
discharge passage 102.
The void 103 provides for the vacant position and reduces
an air-pocket phenomenon, which often occurs at the point of
25 communication between the sample introducing passage 101 and
the air discharge passage 102. The occurrence of the air-
pocket phenomenon results in inaccurate measurements such that
the void 103 ensures accurate and reproducible sampling.
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The ratio of the width of the air discharge passage 102
to that of the sample introducing passage 101 is preferred to
be no more than 1:2. The most preferable range is 1:5 to 1:2.
A ratio below 1:2 ensures the containment of an exact amount
5 of sample in channel 101 with minimal fill over through the
air discharge passage 102.
In Fig. 1, the angle of communication {§) between the
sample introducing passage 101 and the air discharge passage
102 is shown as 90°. But, according to another embodiment of
10 the present invention, this angle may be varied within a
range of from about 45° to about 135°, preferably, from about
75° to about 105°.
As also shown in Fig 1., the void 103 extends beyond the
point of communication from the sample introducing passage 101.
15 To ensure an exact amount of sampling with no bubble formation,
hydrophilic treatment of the sample introducing passage 101
including the void 103 is desired.
The sample introducing part 100 of the present invention
has a capacity to introduce 0.1-3.0 fd of a sample. More
20 preferably, this capacity is 0.1-1.0 fd; most preferably, the
capacity is 0.3-0.7 fd. Samples less than 0.1 fd are too small
to give an accurate measurement within the current biosensor's
range of error. Meanwhile, samples greater than 3.0 fd are
excessive. In preferred embodiments, accurate measurements
25 have been obtained with samples of just 0.5 fit.
Pressing an organic polymer film consisting of polyester,
polyvinyl chloride, or polycarbonate could make the
introduction of the spacer 200 between the base and upper
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substrate. It could be fabricated by pressing a double-sided
adhesive film made of organic polymer, or screen-printing a
layer of adhesive with the pattern shown in Fig. 1.
The working principle of the sample introducing part 100
5 is described in detail as follows.
First, the sample is introduced to the sample
introducing passage 101, by way of capillary action, as soon
as the sample comes into contact with the mouth of the sample
introducing passage 101, and the passage 101 is filled with
10 the sample up to the void space 103. Extra sample is then
forwarded to the air discharge passage 102. Herein, the sample
fill-over can be minimized by controlling the ratio of the
width of the air discharge passage 102 to that of the sample
introducing passage 101 to less than 1:2, and the hydrophilic
15 void 103 removes the air-pocket forming phenomenon occurring
at the point of communication between the sample introducing
passage 101 and the air discharge passage 102.
According to the preferred embodiment of the present
invention, given a 0.5 jbli sample capacity, the sample
20 introducing part 100 fills with blood in about 200 - 2000 ms
depending on the hematocrit level, sample storage conditions,
and the type of anti-coagulant used. Fresh blood samples
normally fills the 0.5 ill sampling channel in about 200 - 8 00
ms as a function of hematocrit level.
25 The sample introducing part 100 of the present invention
may be applied to various types of biosensors, including a
flat type biosensor, a converse type biosensor, a differential
flat type biosensor, a differential converse type biosensor,
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or a converse biosensor with fluidity determining electrode.
Referring to Fig. 2, a flat type biosensor with the
sample introducing part 100 of the present invention
comprises a base substrate 400 on which an electrode system (a
5 working electrode 104 and an reference electrode 105) are
printed, with an oxidase and an electron transfer mediator
immobilized on the electrode system; a sample introducing
spacer 200 having the sample introducing part 100; and an
upper substrate 300 for enclosing the sample introducing parts
10 and for protecting the biosensor from foreign contaminants .
The sample introducing part 100 may be formed as shown, but
the present invention is satisfied as long as the sample
introducing passage 101 communicates with the air discharge
passage 102 and the void 103 is formed at the point of
15 communication; the structure of the void 103 may also be
modified as detailed above.
In the above flat type biosensor, carbon or a conductive
metal material may be printed or deposited on the base
substrate 400 by means of, for example, screen-printing,
20 plasma deposition, or etching to form the working electrode
104 and the reference electrode 105. The two electrodes are
formed symmetrically and extend lengthwise on the base 400.
After the electrode portion is thus constructed, an oxidase
and an electron transfer mediator are spread onto the
25 electrodes.
Except electrode connecting portion, the base substrate
400 is adheres to the sample introducing spacer 200 using an
adhesive. The sample introducing spacer 200 is preferably made
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of insulating polymer, but not limited thereto. The base
substrate 400 and the upper substrate 300 are fixed using
adhesives or a double-sided adhesive tape. Using similar
adhesive means, the fabrication of the biosensor may be
5 completed by pressing the upper substrate 300, serving as a
cover, onto the sample introducing spacer 200.
Fig. 3 illustrates a converse type biosensor with a
sample introducing part 100, characterized in that a base
10 substrate 400' on which a working electrode 104' and an
electrode connector 106 are printed, and an oxidase and an
electron transfer mediator are immobilized on the working
electrode 104' ; a sample introducing spacer 200' having the
sample introducing part 100; and an upper substrate 300' on
15 the bottom side of which an reference electrode 105, and an
electrode connector 106 are printed. The sample introducing
part 100 may be formed as shown, but the present invention is
satisfied as long as the sample introducing passage 101
communicates with the air discharge passage 102 and the void
20 103 is formed at the point of communication; the structure of
the void 103 may also be modified as detailed above.
The fabrication of the converse type biosensor with the
sample introducing part 100 can be accomplished in the same
manner as the flat type biosensor with the sample introducing
25 part 100.
As shown in Fig. 4, a differential flat type biosensor
comprises a base substrate 400a on both surfaces of which a
working electrode 104 and an reference electrode 105 are
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printed and an oxidase and an electron transfer mediator are
provided; a pair of sample introducing spacers 200a and .200b,
each having a sample introducing part 100 , respectively fixed
to upper and lower surfaces of the base substrate 400a; and a
5 pair of cover plates 300a and 300b respectively fixed to outer
surfaces of the sample introducing spacers 200a and 200b. The
sample introducing part 100 may be formed as shown, but the
present invention is satisfied as long as the sample
introducing passage 101 communicates with the air discharge
10 passage 102 and the void 103 is formed at the point of
communication; the structure of the void 103 may also be
modified as detailed above.
As shown in Fig. 5, a differential converse type
biosensor comprises a base substrate 400b on both surfaces of
15 which a working electrode 104 and an electrode connector 106
are printed and an oxidase and an electron transfer mediator
are provided; a pair of sample introducing spacers 200a' and
200b' , each having a sample introducing substrate 100,
respectively fixed to upper and lower surfaces of the base
20 substrate 400b; and a pair of cover plates 300a' and 300b' ,
respectively fixed to outer surfaces of the sample introducing
spacers 200a' and 200b' , on inner sides of which an reference
electrode 105' , and an electrode connector 106 are printed.
The sample introducing part 100 may be formed as shown, but
25 the present invention is satisfied as long as the sample
introducing passage 101 communicates with the air discharge
passage 102 and the void 103 is formed at the point of
communication; the structure of the void 103 may also be
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modified as detailed above.
As shown in Fig. 6, illustrated is a converse type
biosensor with sample fluidity determining capacity,
characterized in that a base substrate 400' on which a working
5 electrode 104', an electrode connector 106, and fluidity
determining electrode 107 are printed, and an oxidase and an
electron transfer mediator are immobilized on the working
electrode 104' ; a sample introducing spacer 200' having the
sample introducing part 100; and an upper substrate 300' on
10 the bottom side of which an reference electrode 105' , and an
electrode connector 106 are printed. The sample introducing
part 100 may be formed as shown, but the present invention is
satisfied as long as the sample introducing passage 101
communicates with the air discharge passage 102 and the void
15 103 is formed at the point of communication; the structure of
the void 103 may also be modified as detailed above. The
fluidity of a sample is determined as a function of sample
filling speed between the first contact point of electrode
105' near the sample introducing mouth and the fluidity
20 determining electrode 107 which is located either at the void
103 or at the air discharge passage 102.
The substrates of any of the base plates or cover plates
for use in the biosensors described above may be made of
25 ceramic, glass, or polymeric materials, with a preference for
an organic polymer of polyester, polyvinyl chloride, or
polycarbonate .
The fabrication of the electrodes, such as the reference
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electrodes, working electrodes, and reference electrodes, may
be achieved using a conductive material, e.g., silver epoxy,
silver/silver chloride, carbon, redox couples, or a modified
conductive carbon paste containing a resin binder. These
5 materials may be formed into reference, counter, and working
electrodes by a screen-printing method, a vapor deposition
method followed by etching, or an adhesion of a conductive
tape .
The above-described biosensors with the sample
10 introducing part 100 have several advantages.
(1) The air-pocket phenomenon, occurring at the point of
communication between the sample introducing passage and air
discharge passage, is eliminated while the sample is rapidly
introduced into the biosensor.
15 (2) As the sample introducing part 100 is well enclosed
by the narrow mouth and air discharge passage, the biosensors
of the present invention maintain a consistent sample
concentration with minimal evaporation, thus improving
analytical reproducibility. In addition, the sample is better
20 contained with the present invention than other types of
sample introducing schemes when the strips are adapted to and
removed from instruments, thereby considerably reducing the
possibility of contamination.
(3) The biosensors equipped with the sample introducing
25 part 100, in which the sample introducing passage and air
discharge passage communicate in a roughly perpendicular
manner, are capable of rapidly introducing a predetermined
amount of sampled blood and increasing accuracy and
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reproducibility. This is in contrast to the conventional i-
type biosensor.
(4) The present invention allows easier blood sampling
through the tip of the biosensor when it is applied to body
5 parts.
The electron transfer mediator provided for the working
electrode may employ ferrocene or its derivatives, quinone or
its derivatives, organic conducting salts, or viologen.
Preferably, the electron transfer mediator is a mixed-valence
10 compound capable of forming redox couples, including
hexaamineruthenium (III) chloride, potassium f erricyanide,
potassium f errocyanide, dimethyl ferrocene, f erricinium,
ferocene-monocarboxylic acid, 7,7,8,8-
tetracyanoquinodimethane, tetrathiaf ulvalene, nickelocene, N-
15 methylacidinium, tetrathiatetracene, N-methylphenazinium,
hydroquinone, 3-dimethylaminobenzoic acid, 3-methyl-2-
benzothiozolinone hydrazone, 2-methoxy-4-allylphenol , 4-
aminoantipyrin , dimethylaniline , 4-aminoant ipyrene , 4 -
methoxynaphthol, 3, 3' , 5, 5' -tetramethylbenzidine , 2, 2-azino-
20 di- [3-ethylbenzthiazoline sulfonate], o-dianisidine, o-
toluidine, 2, 4-dichloro phenol, 4 -aminophena zone , benzidine,
and Prussian blue. Of those, hexaamineruthenium (III)
chloride is a preferred mediator for the proposed biosensor
system, since it satisfies several conditions: (1) both an
25 oxidized and a reduced states thereof in aqueous solution are
stable and reversible; (2) the reduced electron transfer
mediator is non-reactive to oxygen; (3) its formal potential
is low enough to minimize the influence of interfering
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materials such as ascorbic acid, uric acid, and
acetaminophen; (4) the oxidation of the reduced electron
transfer mediator is not sensitive to pH; and (5) it does not
react with electrochemically interfering materials, such as
5 ascorbic acid, acetaminophen, and uric acid.
Herein, it should be understood that the present
invention, although described for biosensors for analysis of
blood glucose levels, can introduce appropriate enzymes and
electron transfer mediators to the electrode system so that a
10 variety of samples, including bio-materials, such as
metabolites, e.g., cholesterol, lactate, creatinine, proteins,
hydrogen peroxide, alcohols, amino acids, and enzymes, e.g.,
GPT (glutamate pyruvate transaminase) and GOT (glutamate
oxaloacetate transaminase) , environmental materials,
15 agricultural and industrial materials, and food materials can
be quantitatively analyzed. For instance, cholesterol,
lactate, glutamate, hydrogen peroxide, and alcohol can be
quantitatively analyzed using glucose oxidase, lactate
oxidase, cholesterol oxidase, glutamate oxidase, horseradish
20 peroxidase, or alcohol oxidase, respectively.
A better understanding of the present invention may be
obtained in light of the following examples which are set
forth to illustrate, but are not to be construed to limit the
present invention .
25
Example 1: Fabrication of a Flat type Biosensor
Conductive carbon paste was screen-printed to form a
symmetrical pattern on a polyester base plate 400 to give
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working electrode 104 and a reference electrode (or reference
electrode) 105. The interval between the two electrodes is
125 \im. A curing of the printed electrodes at 14 0 °C for five
minutes yielded a single electrode body for a flat type
biosensor .
Thereafter, the sample introducing part 100, comprising
the sample introducing passage 101, air discharge passage 102,
and void 103 formed therein, was fixed by pressing double-
sided tape made of polyester. The sample introducing passage
101 communicates perpendicularly with the air discharge
passage 102, and the ratio of the width of the air discharge
passage 102 to that of the sample introducing passage 101 was
controlled to be 1:2. The void 103 was formed to extend
beyond the sample introducing passage 101. The total amount of
sampled blood within the sample introducing part 100 was 0.5
The frame for the biosensor was prepared by inserting to
each polyester base plate 400 and by pressing double-sided
tape made of polyester as a sample introducing spacer 200
having the sample introducing part 100. A solution
containing 0.015 mg of hexaamineruthenium (III) chloride,
0.015 mg of a dispersant (carboxymethylcellulose) , 0.01 mg of
a surfactant (Triton X-100) , and 40 mg of glucose oxidase was
applied to the electrodes for forming the biosensor, and the
resultant deposit was allowed to dry for thirty minutes at
45°C.
Pressing a cover plate 300 onto the sample introducing
spacer 200 completed the flat type biosensor of Fig. 2.
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Example 2: Fabrication of a converse type Biosensor
As shown in Fig. 3, a working electrode 104' and an
5 electrode connector 106 were screen-printed with conductive
carbon paste, and a curing was carried out at 14 0°C for five
minutes. Then, a circuit connector was screen-printed with
the silver paste on one end of the electrode connector 106.
The cover plate with the printed electrode as a reference
10 (auxiliary) electrode 105' was screen-printed with carbon
paste and was cured. Finally, the biosensor was fabricated
such that the end of the reference electrode 105' was screen-
printed with silver paste to be the circuit connector.
The sample introducing spacer 200' comprising the sample
15 introducing passage 101, air discharge passage 102, and void
103 was placed on the base substrate by pressing double-sided
tape made of polyester. The ratio of the width of the air
discharge passage 102 to that of the sample introducing
passage 101 was 1:4, and the total amount of sampled blood
20 within the sample introducing part 100 was adjusted to 0 . 5 f/Jt.
A 1 ml solution containing 0.015 mg of
hexaamineruthenium (III) chloride, 0.015 mg of a dispersant
(carboxymethylcellulose) , 0.01 mg of a surfactant (Triton X-
100) , and 40 mg of glucose oxidase was applied to the
25 electrodes forming the biosensor, and the reaction layer was
allowed to dry for thirty minutes at 4 5°C.
Pressing the cover plate 300' onto the sample
introducing spacer 200' , so as to connect with the circuit
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connector of the base substrate 400' , completed the biosensor
shown in Fig. 3.
Example 3: Fabrication of a Differential Flat type Glucose
5 Sensor
The differential flat type glucose sensor was prepared
in the same manner as in Example 1. As shown in Fig. 4, the
differential flat type biosensor was fabricated by providing
10 a small amount of bovine serum albumin (BSA) on the
differential working electrode 104 of the base substrate 400a,
instead of the hexaamineruthenium (III) chloride and glucose
oxidase used in Example 1, and by pressing the cover plates
300a and 300b.
15
Example 4: Fabrication of a Differential Converse Type
Biosensor
The differential converse type glucose sensor was
20 prepared in the same manner as in Example 2. As shown in Fig.
5, the differential converse type biosensor was fabricated by
providing a small amount of bovine serum albumin (BSA) on the
differential working electrode 104' of the base substrate 400b,
instead of hexaamineruthenium (III) chloride and glucose
25 oxidase used in Example 1, and by pressing the cover plates
300a' and 300b' .
Example 5: Fabrication of Biosensor with Fluidity Determining
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Electrode
The biosensor with fluidity determining electrode was
the converse type biosensor prepared in the same manner as in
5 Example 2 except the use of fluidity determining electrode
107; as illustrated in Fig. 6, it was screen-printed with the
same carbon paste. The tip of the fluidity- determining
electrode was placed at the void 103 of the sample
introducing part.
10
Experimental Example 1: Influence of Interfering Materials on
a Converse Type Glucose Sensor
Figure 7 shows the total response currents to phosphate
15 buffer (pH 7.4) standard solutions containing 177 mg/dL of
glucose and interfering materials whose concentrations are
five times higher than the maximum clinical levels (e.g.,
ascorbic acid 570 \M f acetaminophen 660 \xM r and uric acid 916
|iM) . The total response currents were measured by reading the
20 chronoamperometric response 5 seconds after applying the +0.2
V potential to the working electrode 104' (vs. reference
electrode 105' ) . Samples were introduced into the sample
introducing part 100 of the biosensor fabricated as depicted
in Example 2, and their mean volume was 0.5 |iL . Histograms in
25 Fig. 7 show that the sensors are affected insignificantly by
the presence of interfering materials at an applied potential
of +0.2 V.
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Experimental Example 2: Calibration Curve of a Converse type
Glucose Sensor to Glucose Standard Solutions
The converse type glucose sensor prepared in Example 2
5 was assayed for sensitivity with glucose standard solutions.
The calibration curve thus obtained is depicted in Fig. 8.
In this regard, current values were measured ten times at
each concentration under the electrical field of an applied
potential of 0.2 V with respect to the reference electrode.
10 The amount of samples applied to the sample introducing part
was 0.5 ill and the filling time was no more than 200 ms . The
measurements were performed 2 s after introducing the sample
by applying 0.2 V for three seconds, and the current values
were read in five seconds . The dynamic curves thus obtained
15 are depicted in Fig. 9, wherein the respective curves show
glucose concentrations of Omg/dL (curve a), 50mg/dL (curve b) ,
150mg/dL (curve c) , 30 Omg/dL (curve d) , 450 mg/dL (curve e) ,
and 60 Omg/dL (curve f ) .
Demonstrating that the converse type glucose sensor of
20 the present invention is reliable, the curve was evaluated
and shown to have a slope ( |aA per mg/dL) of 0.093 and
linearity as high as 0.997.
Experimental Example 3: Measurement of the Blood Fluidity
25
The biosensor equipped with fluidity determining
electrode was prepared as described in Example 5. 200 mV of
potential was applied to the working electrode 104' and the
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fluidity determining electrode 107 (vs. the reference
electrode 105' ) . When blood samples are introduced through the
sample introducing passage 101, a sudden change in current is
detected, and the time measurement begins. As soon as the
5 sample reaches the void 103, the second surge of current is
detected and the time interval between the first and second
surge of current is recorded. The relationship between the
sample introducing time and hematocrit level is shown in Fig.
10. The experiment was performed with the NaF treated whole
10 blood containing 180 mg/dL of glucose and varying level of
hematocrit. The fitting equation obtained was Y = -72.23 +
0.58691X - 0. 00084073 X 2 - 1.1211xl0" 6 X 3 + 5.7521xl0" 9 X 4 -
9 . 1172xl0" 12 X 5 , where Y is the estimated hematocrit level from
the sample filling time X measured with the fluidity
15 determining electrode. Table 1 shows the level of hematocrit
estimated from the speed of sample filling time.
Table 1. Hematocrit level estimated from the sample filling
time of the biosensor prepared in Example 5.
20
Hematocrit (%)
Prepared sample
Speed (ms)
Estimated
Hematocrit (%)
30 %
326
30.3 %
35 %
352
32.8 %
40 %
530
41.8 %
45 %
634
44.0 %
50 %
1129
50.1 %
55 %
1791
54.7 %
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In a separate experiment, calibration curves were
obtained with the whole blood at various hematocrit levels
and the relationship between the hematocrit level and the
5 response slopes was formulated (Table 2) .
Hematocrit
Equation (y = current x = glucose)
30 %
y = 0.035934 x - 1.7228
35 %
y = 0.030559 x - 1.31815
40 %
y = 0.025831 x - 1.0137
45 %
y = 0.021752 x - 0.80945
50 %
y = 0.018322 x - 0.7054
55 %
y = 0.015539 x - 0.70155
The correction factors derived in this manner were used
to recalibrate the measured glucose level with respect to the
10 whole blood having 40 % hematocrit level, resulting in the
biosensors that provide hematocrit independent glucose
concentrations. The meter reads the speed of sample
introduction first and determines the level of hematocrit in
the blood sample, looks up the table that provides the
15 corresponding calibration curves, and determines the level of
glucose from the measured currents. Table 3 shows the results
of the experiment carried out as outlined. It is seen that
the hematocrit level correction provides the glucose levels
close to those obtained with YSI 2300.
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Table 3. Glucose concentration in whole blood; the sample
introducing speed measured with the fluidity determining
electrode and the calibration curve in Table 2 were used to
5 estimate the glucose level in whole blood.
Hematocrit %
y > 1 LA V ' \S i»_> v
YSI2300 (mg/dL)
Hematocrit
corrected (mg/dL)
30 %
111
117
202
186
381
392
35 %
138
141
200
207
276
277
40 %
107
112
196
195
266
264
45 %
103
105
190
189
367
363
50 %
102
107
142
143
253
256
55 %
125
144
241
240
332
331
The fluidity determining electrode also discriminate the
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blood samples of unusual fluidity, i.e., samples with too
high or too low hematocrit levels and the fouled introduction
of blood samples due to the formation of air bubble. In such
cases a measuring device may be programmed to issue a warning
5 message or error code for the measurement.
26
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What is claimed is:
1. An electrochemical biosensor with a sample
introducing part, the sample introducing part comprising a
sample introducing passage, an air discharge passage, and a
void, wherein the sample introducing passage communicates with
the air discharge passage and wherein the void is formed at
the point of communication.
2. The electrochemical biosensor according to claim 1,
wherein the ratio of the width of the air discharge passage to
that of the sample introducing passage is no more than 1:2.
3. The electrochemical biosensor according to claim 1,
wherein the ratio of the width of the air discharge passage to
that of the sample introducing passage is in the range of 1:5
to 1:2.
4. The electrochemical biosensor according to claim 1,
wherein the sample introducing part has a capacity to
introduce 0.1-3.0 fd of a sample.
5. The electrochemical biosensor according to claim 1,
wherein the sample introducing part has a capacity to
introduce 0.1-1.0 jut of a sample.
6. The electrochemical biosensor according to claim 1,
wherein the sample introducing part has a capacity to
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WO 03/056345
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introduce 0 . 3-0 .1 {d of a sample.
7. The electrochemical biosensor according to claim 1,
wherein the sample introducing passage communicates with the
5 air discharge passage at an angle of 45-135°.
8. The electrochemical biosensor according to claim 1,
wherein the sample introducing passage communicates with the
air discharge passage at an angle of 75-105°.
10
9. The electrochemical biosensor according to claim 1,
wherein the sample introducing passage communicates with the
air discharge passage at an angle of 90°.
15 10. The electrochemical biosensor according to claim 1,
further comprising an oxidase selected from the group
consisting of glucose oxidase, lactate oxidase, cholesterol
oxidase, glutamate oxidase, horseradish peroxidase, and
alcohol oxidase.
20
11. The electrochemical biosensor according to claim 1,
further comprising an electron transfer mediator selected from
the group consisting of hexaamineruthenium (III) chloride,
potassium f erricyanide, potassium f errocyanide,
25 dimethyl ferrocene, ferricinium, f erocene-monocarboxylic acid,
7,7,8, 8-tetracyanoquinodimethane, tetrathiaf ulvalene,
nickelocene, N-methylacidinium, tetrathiatetracene, N-
methylphenazinium, hydroquinone, 3-dimethylaminobenzoic acid,
28
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3- methyl-2-benzothiozolinone hydrazone, 2 -methoxy- 4 ~
allylphenol, 4-aminoantipyrin, dimethylaniline, 4-
aminoantipyrene, 4 -met hoxynaphthol r 3, 3' ,5,5'-
tetramethylbenzidine, 2 , 2-azino-di- [ 3-ethylbenzthiazoline
sulfonate], o-dianisidine, o-toluidine, 2,4-dichloro phenol,
4- aminophenazone, benzidine and Prussian blue.
12. The electrochemical biosensor according to claim 11,
wherein the electron transfer mediator is hexaamineruthenium
(III) chloride.
13. The electrochemical biosensor according to claim 1,
wherein the biosensor is a flat type biosensor and further
comprises :
a sample introducing spacer;
a base substrate, coupled to said sample introducing
spacer, on a surface of which a working electrode and a
reference electrode are printed and an oxidase and an electron
transfer mediator are provided; and
a cover plate, pressed to said sample introducing spacer,
for forming the sample introducing channel,
wherein the sample introducing part is formed in one end
of said sample introducing spacer.
14. The electrochemical biosensor according to claim 1,
wherein the biosensor is a converse type biosensor and further
comprises :
a sample introducing spacer;
29
WO 03/056345
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a base plate, coupled with said sample introducing spacer,
on a surface of which a working electrode and an electrode
connector are printed and an oxidase and an electron transfer
mediator are provided; and
a cover plate, pressed to said sample introducing spacer,
on inner surface of which a reference electrode and an
electrode connector are printed,
wherein the sample introducing part is formed in one end
of said sample introducing spacer.
15. The electrochemical biosensor according to claim 1,
wherein the biosensor is a differential flat type biosensor
and further comprises:
a pair of sample introducing spacers;
a base plate, coupled between said sample introducing
spacers, on both surfaces of which a pair of working and
reference electrodes are printed, respectively, and an oxidase
and an electron transfer mediator on one side, and BSA and an
electron transfer mediator on the other side are provided,
respectively; and
a pair of cover plates are pressed to both surfaces of
said sample introducing spacers for forming the sample
introducing channels ,
wherein the sample introducing parts are formed in one
end of each of said sample introducing spacers.
16. The electrochemical biosensor according to claim 1,
wherein the biosensor is a differential converse type
30
WO 03/056345
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biosensor and further comprises:
a pair of sample introducing spacers;
a base plate, coupled between said sample introducing
spacers, on both surfaces of which a working electrode is
5 printed, respectively, and an oxidase and an electron transfer
mediator on one side, and BSA and an electron transfer
mediator on the other side are provided, respectively; and
a pair of cover plates having reference electrode and
electrode connection part on inner surface are pressed to both
10 said sample introducing spacers to form the sample introducing
channels,
wherein the sample introducing parts are formed in one
end of each of said sample introducing spacers.
15 '17. The electrochemical biosensor according to claim 1,
wherein the biosensor is a converse type biosensor and further
comprises :
a sample introducing spacer;
a base plate, coupled with said sample introducing spacer,
20 on a surface of which a working electrode and an electrode
connector, and a fluidity determining electrode are printed,
and an oxidase and an electron transfer mediator are provided;
and
a cover plate, pressed to said sample introducing spacer,
25 on inner surface of which a reference electrode and an
electrode connector are printed,
wherein the sample introducing part is formed in one end
of said sample introducing spacer.
31
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1/10
FIG. 1
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2/10
FIG. 2
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3/10
FIG. 3
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5/10
FIG. 5
^105' 106
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6/10
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7/10
FIG. 7
12-1
10
8
% 6
2H
0
abed
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9/10
FIG. 9
| i i i i | i i i i | i m — | i / 7 / i t i t t | i i
0 1 2 3 4 5
Time.sec
WO 03/056345
PCT/KR02/00703
10/10
FIG. 10
2000-
1800-
1600-
1400-
w
B 1200
a>
£ 1000-
800
600-
400
200 -I
n — r
28
32
n — r
38
— i — 1 — r~
40 44
Hematocrit „ %
n — r
48
52 56
n — r
60
INTERNATIONAL SEARCH REPORT
' WFternational application No.
PCT/KR02/00703
A. CLASSIFICATION OF SUBJECT MATTER ~~ ~~~ - — —
IPC7 G01N 35/10
According to International Patent Classification (IPC) or to both national classification and IPC
~B. FIELDS SEARCHED
Minimum documentation searched (classification system followed by classification symbols) — — -
IPC7G01N 33/48, G01N 27/16, C12Q 1/54
Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched
Korean Patents and applications for inventions since 1975, Korean Utility models and applications for Utility models since 1975
Japanese Utility models and application for Utility models since 1975
Electronic data base consulted during the intertnational search (name of data base and, where practicable, search terms used)
KIPASS
C. DOCUMENTS CONSIDERED TO BE RELEVANT
Category*
Citation of document, with indication, where appropriate, of the relevant passages
Relevant to claim No.
A
US 5,798,031 A (Steven C. Charlton) Aug.25, 1998
- see the whole document -
1-17
A
US 5,562,770 A(GJohn Pritchard) Jun.9, 1998
- see the whole document -
1-17
A
US 6,270,637 Bl(William RCrismore) Aug.7, 2001
- see the whole document -
1-17
A
WO 97/02487 Al (BOEHRINGER MANNHEIM Corp) Jan.23, 1997
- see the whole document -
1-17
| ] Further documents are listed in the continuation of Box C.
□
See patent family annex.
"A"
"E"
"O"
Special categories of cited documents: " "j"
document defining the general state of the art which is not considered
to be of particular relevence
earlier application or patent but published on or after the international »x n
filing date
document which may throw doubts on priority claim(s) or which is
cited to establish the publication date of citation or other "y"
special reason (as specified)
document referring to an oral disclosure, use, exhibition or other
means
document published prior to the international filing date but later »&»
than the priority date claimed
later document published after the international filing date or priority
date and not in conflict with the application but cited to understand
the principle or theory underlying the invention
document of particular relevence; the claimed invention cannot be
considered novel or cannot be considered to involve an inventive
step when the document is taken alone
document of particular relevence; the claimed invention cannot be
considered to involve an inventive step when the document is
combined with one or more other such documents, such combination
being obvious to a person skilled in the art
document member of the same patent family
Date of the actual completion of the international search
19 SEPTEMBER 2002 (19.09.2002)
Date of mailing of the international search report
19 SEPTEMBER 2002 (19.09.2002)
Name and mailing address of the ISA/KR
jgP|^ Korean Intellectual Property Office
m ^;W't 920 Dunsan-dong, Seo-gu, Daejeon 302-701,
^Ljg Republic of Korea
Facsimile No. 82-42-472-7 1 40
Authorized officer ^^^^ ^
JOO ; Young Sik \ • ,
Telephone No. 82-42-481-5995 " ^ ^-^j*
Form PCT/ISA/210 (second sheet) (July 1998)
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